Genotype Mutations in Palestinian Children with Familial Mediterranean Fever: Clinical Profile, and Response to Colchicine Treatment: A Retrospective Cohort Study

Background: Familial Mediterranean fever is a hereditary autoinflammatory disease affecting mainly Arabs, Turks, Armenians, and Jews with genotype-phenotype heterogeneity, presenting as recurrent episodes of fever along with polyserositis and rash. To date, more than 370 mutations in the MEFV gene have been recognized to cause the disease. Methods: We conducted a retrospective cohort study involving 124 patients in Hebron, Palestine, diagnosed with FMF at the Al-Ahli, and Palestinian Red Crescent Society (PRCS) Hospitals. Results: The median age of diagnosis was five years, presenting as abdominal pain (76.6%), fever (67.7%), joint pain and arthritis. Regarding MEFV gene mutations, we had 62 patients (50%) with heterozygous genotypes, 40 patients (32.3%) with homozygous phenotypes, 21 patients (16.9%) with compound heterozygous genotypes, and one was a missing state. Regarding variant frequencies, M694V was the most common one (43.4%), followed by E148Q (15.6%), V726A (5.7%), A744S (4.1%), and R202Q (4.1%). Positive family history was detected in 59 patients (54.6%), and there was no significant difference in zygosity regarding characteristics, consanguinity, and family history. Conclusions: We affirm in this study of 124 children with FMF, abdominal pain, followed by fever, joint pain and arthritis were the main manifestations. Further, M694V, E148Q, V726A, A744S, and R202Q were the most frequent mutations, and carrying the M649V mutations is associated with a predisposition to other comorbidities. We believe that this study gives a pervasive overview of FMF in Palestinian patients. Looking forward, future studies on a larger number of patients could precisely highlight the genotype-phenotype association among FMF patients.


INTRODUCTION
Familial Mediterranean fever (FMF) is considered the most common inherited monogenic autoinflammatory periodic fever condition.FMF is additionally known as 'periodic peritonitis'.2] FMF usually follows an autosomal recessive inheritance pattern which means that both alleles (homozygosity) should be mutated.Nevertheless, many patients have been diagnosed with FMF while having only one genetic mutation (heterozygosity) with significant response to the treatment. 3This genetic abnormality results in gaining of a new function in the MEFV gene. 46] Episodic complaints begin before the age of 20, with each episode lasting from several hours to a few days.Headache is an accompanying complaint besides fever. 7he most recent criteria were published in 2020 entitled 'Eurofever/PRINTO Classification Criteria', which combines clinical manifestation and genetic finding together, making it more sensitive (96%) than Tel Hashomer (88.4%) and Yalcinkaya-Ozen criteria (93.4%), but less specific.According to Eurofever/PRINTO Classification Criteria, diagnosis of FMF can be established by the presence of confirmatory MEFV genotype and at least one of the following: (1) duration of episodes 1-3 days, (2) Chest pain, (3) Arthritis, (4) Abdominal pain or presence of non-confirmatory MEFV genotype and at least two of the following: (1) Duration of episodes 1-3 days, (2) Chest pain, (3) Arthritis, (4) Abdominal pain. 8enetic and molecular analysis plays a fundamental role in establishing the diagnosis of clinically suspected FMF patients.The MEFV gene is located on the short arm of chromosome 16 and is considered the responsible gene in FMF. 9 Approximately 370 variants of the MEFV gene have been discovered.Among these sequence variants, mutations in the M694I, M694V, E148Q, V726A, and M680I genes are responsible for the vast majority of FMF cases.][12][13] Since the 1970s, Colchicine has been considered the treatment of choice for FMF. 14The main goal of colchicine administration is to decrease and prevent the attacks frequency, making the patient symptoms-free between the episodes, and to prevent and halt amyloidosis development and progression. 15The severity of symptoms and treatment response are strongly correlated to the genetic variant, with the homozygous M694V mutation tend to have very severe disease and more colchicine resistance. 16his study aims to detect the genetic background of FMF, determine the most frequent MEFV mutations among Palestinians, and evaluate the clinical profile and the response to colchicine treatment with different genetic variants.

MATERIALS AND METHODS
The current study is a double-centre, retrospective cohort survey that included 124 children and adolescents of both sexes (≤18 years) with the ascertained diagnosis of FMF according to their clinical manifestations and molecular basis, throughout their routine follow-up visits at the Rheumatology Outpatient Clinics of the paediatric department, Al-Ahli and Palestinian Red Crescent Society (PRCS) hospitals, Hebron, Palestine.The study was carried out during the last two weeks of May 2022.This study aims to investigate the genetic background of FMF among our included patients and identify the most frequent MEFV genetic mutations, to study how different genetic mutations could affect the phenotypic expression of the disease, to detect the most frequent presenting manifestation, and to evaluate patient's response to colchicine treatment with different genetic variants.Diagnosis of FMF was established referring to the most recent criteria published in 2020 entitled 'Eurofever/ PRINTO Classification Criteria' which combines clinical manifestation and genetic findings together.Patients who did not meet the criteria were excluded from the study.All patients had a comprehensive personal and family history, physical and laboratory assessment, and after obtaining the approval to conduct this study, authors electronically reviewed and validated the data from the medical records.In case of incomplete or missing data, patients were reevaluated.The outcomes of MEFV molecular analysis were documented.Subsequently, our patients were divided into either having homozygous (both alleles of the gene are affected), heterozygous (only one allele of the gene is affected) or compound heterozygous (mutated alleles on different MEDITERRANEAN JOURNAL OF RHEUMATOLOGY 34 3 2023 genes).Genetic analysis was performed, to recognize the 11 most prevalent MEFV mutations; the M694V, M694I, E148Q, R202Q, M680I, R329H, V726A, P369S, SCN9A, A744S and A408G.DNA was extracted from blood using QIAamp DNA mini kit followed by FMF targeted variants detection using FMF Multiplex real time PCR kit (Cat.No: 11R-20-13) from SNP Biotechnology.

Data collection process
Medical records of patients were collected electronically from the hospital system, while some patients were faceto-face interviewed and re-evaluated.

Statistical analysis
We evaluated the normality of the distribution of quantitative variables using the Shapiro-Wilks test.Data were statistically described in terms of median and interquartile range for quantitative variables with nonnormal distribution, and frequency and percentage for qualitative variables.In the absence of normal distribution, nonparametric tests used for the Comparison of numerical variables between the study groups were done using the Kruskal Wallis test followed by post hoc pairwise comparisons between groups using the Bonferroni adjustment method after significant effects have been found for comparing categorical data, the Chi-square (χ2) test was performed.A P-value less than 0.05 was considered statistically significant.We did all statistical calculations using R language programming software version 4.1.2and Microsoft Excel 365.

RESULTS
The omitted data has been presented in Table 1 to elucidate any discrepancies or incongruities with the data presented in other tables.We identified 124 cases with familial Mediterranean fever and according to gene mutations and allele status, we classified our patients into three groups: the first group (homozygous group) included 40 patients of whom 16 (40%) were males and 24 (60%) were females, second group (heterozygous group) included 62 patients of whom 27 (43.5%)were males and 35 (56.5%) were females and third group (Compound Heterozygous) included 21 patients of whom 12 (60%) were males and 8 (40%) were females.The median age [IQR] of our population and median age at diagnosis of all study populations were 9.00 [6.00, 13.00], and 5.00 [3.00, 8.00] years respectively.Comparing characteristics, consanguinity, and family history between different genetic zygosity, none of the parameters was statistically significant.Positive family history was reported in a higher percentage among Heterozygous 30 (56.6%), which was not significant.Most of our patients received colchicine therapy (n= 92, 74%); with a median dose of 1.00 [0.50, 1.00] mg/day, and 64 (69.6%) of our patients reported improvement.Homozygous and compound heterozygous patients were more likely to receive a higher dose of colchicine than heterozygous patients (median dose 1 vs. 0.88 mg/ day) (Table 2).Regarding the clinical features, abdominal pain was the most common presenting feature in 95 (76.6%) cases, followed by fever in 84 (67.7%).Joint pain and arthritis were common in both heterozygotes and the homozygotes groups and were documented in 16 (25.8%)and 17 (42.5%)patients, respectively (Figure 1).Regarding the consanguinity of the study population, 7 (6.3%) had consanguineous parents; 3 (5.3%)were heterozygous; 2 (5.7%) were homozygous, and 2 (11.1%) were compound heterozygous.We recorded a positive family history of FMF in 59 patients (54.6 %), of whom 30 (56.6%) were heterozygous and 19 (51.4%) were homozygous.The most common seven mutations in our patients were (M694V, E148Q, V726A, A744S, R202Q, M694I, M694V + V726A, and M694V + R202Q).M694V was the most prevalent variant in the overall cohort 53 (43.4%)The M694V genotype distribution was homozygous in 32 (82.1%) and heterozygous in 21 (33.9%).The mutation E148Q was identified in 19 cases (15.6%), and all of these instances were found to be in a heterozygous state (30.6%) (Table 3).FMF was associated with other disorders such as: juvenile idiopathic arthritis (JIA) in 3 patients having the M694V mutation; severe form of Henoch-Schoenlein purpura (HSP) was found in one patient having the M694V mutation; osteomyelitis was found in one patient having the V726A and E148Q      4).
We employed Direct DNA sequencing to examine the exons of all individuals diagnosed with FMF, aiming to identify any mutations potentially responsible for the disease.Among the FMF patients, 80 exhibited an exon 10 mutation, primarily involving four variants: M694V, M694I, V726A, and M680I, which collectively accounted for the majority of cases.In addition, we detected 27 cases with mutations outside of exon 10.Notably, 15 individuals displayed a combination of mutations, encompassing both exon-10 and non-exon-10 mutations.According to laboratory findings, elevated inflammatory markers were noticed more apparently in those with exon-10 mutations   than others as outlined in Table 5.When reviewing the physical examination records, instances of abdominal pain were most frequently reported in the exon 10 mutant group (62 cases, 77.5%), followed by the non-exon mutation group (22 cases, 81.5%), and the mixed group (10 cases, 66.7%).8] Although this disease affects Arab societies, there is no sufficient data regarding the prevalence of FMF in Arab countries, including Palestine, where the study was done. 19In this study, we aim to identify the most frequent MEFV mutations as well as to evaluate the clinical profile and response to colchicine treatment in patients with genetic variance.Our analysis involved 124 paediatric patients, 67 female (54.5%) and 56 male (45.5%), diagnosed with FMF and positive MEFV mutations.In agreement with Talaat et al. and Duşunsel et al. [20][21] female-to-male ratio was found to be high (1.2:1) compared to other studies that reported a male predominance with a male-to-female ratio of 1.2: 1 in Turkish populations, [22][23] or equal prevalence between Arab males and females. 246 Surprisingly, two FMF patients in this study were diagnosed before the age of one year, which was challenging due to the lack of verbal milestones ex- pressing their complaints on top of the ambiguous presentation.Hence, diagnosing FMF in younger paediatric age groups, particularly in endemic regions, is crucial to decrease late comorbidities. 27arious genotypes are reflected in FMF patients with different ethnicities.To date, more than 370 MEFV genes are involved in the development of the disease. 13n the Middle East, M694I, M694V, V726A, E148Q, and M680I gene variants are responsible for most FMF cases, and the allele frequency for these genes is highly variable even between the same country. 12Our study has revealed M694V (43.4%) as the most frequent mutation, followed by E148Q (15.6%),V726A (5.7%), A744S (4.1%), R202Q (4.1%), M694I (3.3%), and M694V + V726A (3.3%).This somehow corresponds to a survey conducted on a Lebanese population with the same  TITLE mutational profile but with different frequencies, where M694V (20.6%) was found to be the most common mutation, followed by E148Q (17.9%) and V726A (16.0%).R202Q (12.6%),M694I (11.8%), and A744S (10.7%). 28imilarly, previous reports in various Arab countries, including Syria, Jordan, Morocco, and Tunisia, [29][30][31][32][33] found that the M694V variant was the predominant genotype among FMF patients.
Compared with a previous Palestinian study, 34 we discovered that M694V was the leading mutation, followed by V726A, M694I, E148Q, and M680I mutations.However, there were some discrepancies in frequencies; for example, the E148Q mutation, which was the second most prevalent variant in our study (15.6%), was found to be the fourth most prevalent variant in the Palestinian study (8.5%). 34Another notable point is that the R202Q genotype among Arabs was only documented in our study and another study conducted on Lebanese patients.Further, the same R202Q genotype was reported in a study done by Coşkun et al. and Gunesacar et al.
] A positive family history of FMF was documented in 59 patients (54.6 %), of whom 30 were heterozygous, and 19 were homozygous.These findings appear to be high when compared to studies performed by Jarjoure and Al Berrawi, 30 and Talaat et al. 20 who detected a positive family history in 32 (31 %) out of their 103 Syrian patients, and 21 (22.11%)out of their 95 Egyptian patients, respectively.While parental consanguinity was evident in 6.3 % of our study population, this had no effect on genetic zygosity.However, Jarjoure and Al Berrawi, 30 and Talaat et al. 20 reported a higher consanguinity rate in their population as (39.8 %), and 37.89%, respectively.][41] Approximately 7.25% of FMF patients in this study had other concomitant diseases, corresponding to a study done by Ayaz et al. 42 This could be attributed to the persistence of a subclinical inflammatory state between the attacks, 22,[40][41][42][43][44] promoting generalized inflammation and triggering the onset of other auto-inflammatory diseases.
Our results indicated that FMF was associated with JIA in 3 patients (2.41%), Crohn's disease in 3 patients (2.41%), a severe form of HSP in one patient (0.8%), both JIA and ankylosing spondylitis in one patient (0.8%) and hypothyroidism in one patient (0.8%).Carrying exon 10 mutations, such as the M694V mutation, appears to predispose FMF patients to the development of certain inflammatory diseases, which was consistent with our findings.According to Yıldız et al., 18.9% of their Turkish FMF patients have a co-existing condition, the most prevalent of which were uveitis (n = 12, 1.7%), asthma (n=29, 4.2%), JIA (n=42, 6.1%), IgA vasculitis (n = 20, 2.9%), and inflammatory bowel disease (n=10, 1.4%).However, only IBD and asthma patients had the MEFV gene mutation. 46In contrast to our results, a study among 95 Egyptian patients mentioned that 13.68% of their patients had other inflammatory conditions such as JIA (n=4, 4.21%), HSP in (n=3, 3.16%), SLE (n=3,3.16%),and IBD (n=2, 2.11%), while the most frequent correlated mutation was M680I followed by E148Q, V726A and M694V mutations. 20bout 26.9% of our patients had microcytic anaemia, while anaemia in FMF patients can be functional, due to chronic inflammation, absolute iron deficiency (AID), or a combination of both, differentiating between them is essential, as iron therapy alone is adequate to improve anaemia in FMF patients with AID, but not in the other types. 47Further, Celkan et al. 28 mentioned that Colchicine therapy has a role in the treatment of anaemia via suppression of the active inflammatory process.
Regarding the clinical presentation of FMF, abdominal pain is the known presenting complaint among Arab patients. 250 Joint pain and arthritis were a shared feature in both heterozygotes and homozygotes group, and were documented in 16 (25.8%),and 17 (42.5%)patients, respectively.However, fever was the most common complaint among 109 Egyptian patients (96.84%) in a study done by Talaat et al. 20 followed by abdominal pain (94.74%), and arthritis (78.95%).
Our aim regarding treatment is to prevent recurrent attacks, reduce the inflammatory condition and minimise the risk of developing late complications.Since 1972, colchicine has been the cornerstone of treatment due to its effectiveness in reducing the number and the severity of attacks as well as preventing complications such as amyloidosis. 15,22,49The European Alliance of Associations for Rheumatology (EULAR) guidelines recommend a dose of: 0.5 mg/day for children under 5 years of age, 0.5-1.0mg/day in children 5-10 years: and 1.0-1.5 mg/ day for children >10 years and adults. 50 Compound Heterozygous; 17 (68%) were heterozygotes, and 14 (66.7%) were homozygotes.Similarly, an Egyptian study conducted among 70 patients revealed that the required dose for attacks control was significantly lower in heterozygotes than homozygotes, with a better response to colchicine in the heterozygous group. 51e recognise that there are some limitations to our study.First, the small sample size as we cannot find data consistent with ours in terms of detailed clinical features, diagnosis, and treatment at other hospitals.Second, the exclusion of many patients diagnosed with FMF due to incompliance with the therapeutic regimen and follow-up visits.
Eventually, prospective studies on larger scales are needed to screen for other unique and newly addressed variants as well as scrutinise those of ambiguous significance in order to obtain a more precise diagnosis of FMF and thus provide patients with individualised treatment and enhanced disease management outcomes.

CONCLUSION
M694V, E148Q, V726A, A744S, and R202Q were the most common mutations in our patients.Specifically, M694V was the most prevalent variant in the overall cohort.Approximately half of our patients were found to a have heterozygote state.Further, Abdominal pain was the presenting feature among our patients, while Fever, joint pain, and arthritis were also observed as clinical manifestations.FMF syndrome may be associated with other autoimmune diseases as a result of the persisting inflammatory state.Regardless of our current FMF knowledge, we recommend further investigation of a larger number of patients in order to further clarify the genotype-phenotype correlation among Palestinian FMF paediatric patients.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE
All methods were conducted in accordance with the ethical standards of the declaration of Helsinki / Accordance with relevant guidelines and regulations.Ethical approval for this study was sought from the Palestinian Ministry of Health for data collection and also from the Research Ethics Committee -faculty of medicine, Al-Quds University.The objectives of the study were clarified to the participants Confirming that their contribution is completely voluntary and will not affect any treatment or health services that participants or their relatives receive.The data were collected anonymously without subjects or gender identification.All responses were encoded, used primarily for statistical analysis, and treated confidentially.According to the culture and traditions where the study was conducted, there were no social risks or harm.Before reviewing of the patient's medical records, written informed consents were obtained from the participants, the parents/guardians of the minors (minors are considered anyone under the age of 16), and also from legal guardians of the illiterate participants.Verbal informed consent to participate was also obtained from the illiterate participants.

Figure 1 .
Figure 1.Clinical features of all patients of the different genetic zygosity.
values less than 0.05 are considered statistically significant.

Table 2 .
Demographic data showing differences between homozygotes, heterozygotes, and compound heterozygotes.
*P-value less than 0.05 is considered statistically significant; post-hoc testing using Bonferroni adjustment showed that the significance was between heterozygous and homozygous only (p=0.022).IQR: interquartile range; n: number of patients.

Table 3 .
Gene mutations and Alleles analysis in FMF patients.

Table 4 .
Demographic and clinical data, and response to colchicine in the most frequent mutations detected. 3)